Lactic acid bacteria and bifidobacteria for treating endotoxemia

ABSTRACT

The invention relates to use of a bacterium selected from a lactic acid bacterium, a  Bifidobacterium  or a mixture of any thereof for treating metabolic endotoxemia, inhibiting bacterial translocation and regulating lipid absorption in a mammal.

CLAIM FOR PRIORITY

This application claims priority under 35 U.S.C. 371 to InternationalApplication No. PCT/IB2010/053482, filed on Jul. 30, 2010, which claimspriority to U.S. Patent Application No. 61/229,980, filed Jul. 30, 2009,U.S. Patent Application No. 61/312,400, filed Mar. 10, 2010, U.S. PatentApplication No. 61/321,949, filed Apr. 8, 2010, and U.S. PatentApplication No. 61/325,919, filed Apr. 20, 2010, each of which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to new uses of lactic acid bacteria andBifidobacteria, (particularly, although not exclusively, probioticbacteria), and to food products, feed products, dietary supplements andpharmaceutical formulations containing them.

BACKGROUND TO THE INVENTION

Endotoxemia is defined as the presence of an elevated level oflipopolysaccharides (also known as endotoxins) in the body.Lipopolysaccharides (LPS), also known as lipoglycans, are largemolecules consisting of at least one lipid moiety and at least onepolysaccharide moiety joined by a covalent bond. LPS are found in theouter membrane of Gram-negative bacteria, act as endotoxins and elicitstrong immune responses in animals.

Cani et al., Diabetes, 2007, 56, 1761-1772, describes the induction ofan increase in endotoxemia in mice fed a high-fat diet. The authorsfound that plasma LPS concentration varies throughout the day,increasing to a maximum at the end of the dark, feeding period for micefed a normal diet, and that a high-fat diet caused endotoxemia to behigh throughout the day. The authors define the term ‘metabolicendotoxemia’ as a chronic, 2- to 3-fold increase in plasmalipopolysaccharides (LPS) concentration from baseline levels, induced bya high-fat diet, and note that the endotoxemia levels reached was 10-50times lower than that obtained during septic shock.

Bacterial translocation is defined as the passage of viable bacteriafrom the intestinal tract through the epithelial mucosa into the body.Bacteria may enter the lymphatic system via mesenteric lymph nodes andtherefore may be circulated systemically. Bacteria can also enter bloodcirculation (bacteremia) and may also be located in tissues. Bacterialtranslocation may occur in a number of medical conditions, includingintestinal bacterial overgrowth, intestinal injury and shock. Anymedical condition associated with increased intestinal permeability canpotentially lead to bacterial translocation.

As LPS originate from bacteria in the gut, translocation of bacteriafrom the gut into the body may potentially serve as a potentialmechanism for endotoxemia, including metabolic endotoxemia. IfGram-negative bacteria translocate into the body, they serve as a sourceof LPS. However, the exact route of LPS into the body in metabolicendotoxemia is currently unknown: bacterial translocation is consideredone possible explanation, but free LPS from the gut may also enter thebody during normal lipid absorption. It is also possible that severalmechanisms take place at the same time.

Schiffrin et al., Br. J. Nutr., 2009, 101, 961-966, describe the use ofa probiotic yogurt supplement in elderly patients with small-intestinalbacterial overgrowth (SIBO). The effects on intestinal colonisation, gutpermeability, endotoxin translocation and modification of innate immunefunctions were assessed. However, the endotoxemia in the patientsdescribed in this document is septic shock endotoxemia, which is causedby infection with pathogens, such as pathogenic bacteria, and in which,as described in the Cani et al. article referred to above, plasma LPSlevels are increased by a much larger factor from normal levels. This isdistinct from metabolic endotoxemia, which as described above isgenerally caused by diet (in particular, by a high-fat diet) and inwhich the increase in plasma LPS levels (expressed as a multiple ofnormal levels) is much lower.

SUMMARY OF THE INVENTION

In one aspect, the invention provides use of a bacterium selected from alactic acid bacterium, a Bifidobacterium or a mixture of any thereof inthe manufacture of a food product, dietary supplement or medicament fortreating metabolic endotoxemia in a mammal.

In another aspect, the invention provides use of a bacterium selectedfrom the species Lactobacillus acidophilus, Lactobacillus plantarum,Bifidobacterium animalis, Bifidobacterium lactis, or Bifidobacteriumbifidium, or a mixture of any thereof in the manufacture of a foodproduct, dietary supplement or medicament for treating endotoxemia in amammal.

In a further aspect, the invention provides use of a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof in the manufacture of a food product, dietary supplement ormedicament for inhibiting bacterial translocation in a mammal.

In a still further aspect, the invention provides use of a bacteriumselected from a lactic acid bacterium, a Bifidobacterium or a mixture ofany thereof in the manufacture of a food product, dietary supplement ormedicament for treating infections caused by Gram-negative bacteria,treating overgrowth of Gram-negative bacteria, or treating an imbalanceof intestinal and/or mucosal Gram-negative bacteria (in particular inthe gastrointestinal tract) in a mammal.

In a yet further aspect, the invention provides use of a bacteriumselected from a lactic acid bacterium, a Bifidobacterium or a mixture ofany thereof in the manufacture of a food product, dietary supplement ormedicament for treating translocation of Gram-negative bacteria in amammal.

In a still further aspect, the invention provides use of a bacteriumselected from a lactic acid bacterium, a Bifidobacterium or a mixture ofany thereof in the manufacture of a food product, dietary supplement ormedicament for reducing the elevated adhesion of Gram-negative orpathogenic bacteria to the gastrointestinal mucosa of a mammal.

In a yet further aspect, the invention provides use of a bacteriumselected from a lactic acid bacterium, a Bifidobacterium or a mixture ofany thereof in the manufacture of a food product, dietary supplement ormedicament for treating endotoxemia in a mammal by reducing the elevatedadhesion of Gram-negative or pathogenic bacteria to the gastrointestinalmucosa of said mammal.

In a still further aspect, the invention provides use of a bacteriumselected from a lactic acid bacterium, a Bifidobacterium or a mixture ofany thereof in the manufacture of a food product, dietary supplement ormedicament for reducing the elevated adhesion oflipopolysaccharide-containing bacteria to the gastrointestinal mucosa ofa mammal.

In a yet further aspect, the invention provides use of a bacteriumselected from a lactic acid bacterium, a Bifidobacterium or a mixture ofany thereof in the manufacture of a food product, dietary supplement ormedicament for treating endotoxemia in a mammal by reducing the elevatedadhesion of lipopolysaccharide-containing bacteria to thegastrointestinal mucosa of said mammal.

In a further aspect, the invention provides use of a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof in the manufacture of a food product, dietary supplement ormedicament for regulating lipid absorption in a mammal.

In a still further aspect, the invention provides use of a bacteriumselected from a lactic acid bacterium, a Bifidobacterium or a mixture ofany thereof in the manufacture of a food product, dietary supplement ormedicament for treating endotoxemia in a mammal by regulating lipidabsorption in said mammal.

In a still further aspect, the invention provides use of a bacteriumselected from a lactic acid bacterium, a Bifidobacterium or a mixture ofany thereof in the manufacture of a food product, dietary supplement ormedicament for treating bacteremia in a mammal.

In a yet further aspect, the invention provides use of a combination of:

-   (a) a lactic acid bacterium, a Bifidobacterium or a mixture of any    thereof; and-   (b) a prebiotic;-   in the manufacture of a food product, dietary supplement or    medicament for treating endotoxemia in a mammal.

In a yet further aspect, the invention provides a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof for use in treating metabolic endotoxemia in a mammal.

In a still further aspect, the invention provides a bacterium selectedfrom the species Lactobacillus acidophilus, Lactobacillus plantarum,Bifidobacterium animalis, Bifidobacterium lactis, or Bifidobacteriumbifidium, or a mixture of any thereof for use in treating endotoxemia ina mammal.

In a yet further aspect, the invention provides a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof for use in inhibiting bacterial translocation in a mammal.

In a still further aspect, the invention provides a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof for use in treating infections caused by Gram-negative bacteria,treating overgrowth of Gram-negative bacteria, or treating an imbalanceof intestinal and/or mucosal Gram-negative bacteria (in particular inthe gastrointestinal tract) in a mammal.

In a yet further aspect, the invention provides a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof for use in treating translocation of Gram-negative bacteria in amammal.

In a still further aspect, the invention provides a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof for use in reducing the elevated adhesion of Gram-negative orpathogenic bacteria to the gastrointestinal mucosa of a mammal.

In a yet further aspect, the invention provides a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof for use in treating endotoxemia in a mammal by reducing theelevated adhesion of Gram-negative or pathogenic bacteria to thegastrointestinal mucosa of said mammal.

In a still further aspect, the invention provides a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof for use in reducing the elevated adhesion oflipopolysaccharide-containing bacteria to the gastrointestinal mucosa ofa mammal.

In a yet further aspect, the invention provides a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof for use in treating endotoxemia in a mammal by reducing theelevated adhesion of lipopolysaccharide-containing bacteria to thegastrointestinal mucosa of said mammal.

In a further aspect, the invention provides a bacterium selected from alactic acid bacterium, a Bifidobacterium or a mixture of any thereof foruse in regulating lipid absorption in a mammal.

In a still further aspect, the invention provides a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof for use in treating endotoxemia in a mammal by regulating lipidabsorption in said mammal.

In a yet further aspect, the invention provides a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof for use in treating bacteremia in a mammal.

In a yet further aspect, the invention provides a combination of:

-   (a) a lactic acid bacterium, a Bifidobacterium or a mixture of any    thereof; and-   (b) a prebiotic;-   for use in treating endotoxemia in a mammal.

In a still further aspect, the invention provides a method of treatingmetabolic endotoxemia in a mammal in need thereof, the method comprisingadministering an effective amount of a bacterium selected from a lacticacid bacterium, a Bifidobacterium or a mixture of any thereof.

In a yet further aspect, the invention provides a method of treatingendotoxemia in a mammal in need thereof, the method comprisingadministering an effective amount of a bacterium selected from thespecies Lactobacillus acidophilus, Lactobacillus plantarum,Bifidobacterium animalis, Bifidobacterium lactis, or Bifidobacteriumbifidium, or a mixture of any thereof.

In a yet further aspect, the invention provides a method of inhibitingbacterial translocation in a mammal in need thereof, the methodcomprising administering an effective amount of a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof.

In a still further aspect, the invention provides a method of treatinginfections caused by Gram-negative bacteria, treating overgrowth ofGram-negative bacteria, or treating an imbalance of intestinal and/ormucosal Gram-negative bacteria (in particular in the gastrointestinaltract) in a mammal in need thereof, the method comprising administeringan effective amount of a bacterium selected from a lactic acidbacterium, a Bifidobacterium or a mixture of any thereof.

In a yet further aspect, the invention provides a method of treatingtranslocation of Gram-negative bacteria in a mammal in need thereof, themethod comprising administering an effective amount of a bacteriumselected from a lactic acid bacterium, a Bifidobacterium or a mixture ofany thereof.

In a still further aspect, the invention provides a method of reducingthe elevated adhesion of Gram-negative or pathogenic bacteria to thegastrointestinal mucosa in a mammal in need thereof, the methodcomprising administering an effective amount of a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof.

In a yet further aspect, the invention provides a method of treatingendotoxemia in a mammal in need thereof by reducing the elevatedadhesion of Gram-negative or pathogenic bacteria to the gastrointestinalmucosa of said mammal, the method comprising administering an effectiveamount of a bacterium selected from a lactic acid bacterium, aBifidobacterium or a mixture of any thereof.

In a still further aspect, the invention provides a method of reducingthe elevated adhesion of lipopolysaccharide-containing bacteria to thegastrointestinal mucosa of a mammal in need thereof, the methodcomprising administering an effective amount of a bacterium selectedfrom a lactic acid bacterium, a Bifidobacterium or a mixture of anythereof.

In a yet further aspect, the invention provides a method of treatingendotoxemia in a mammal in need thereof by reducing the elevatedadhesion of lipopolysaccharide-containing bacteria to thegastrointestinal mucosa of said mammal, the method comprisingadministering an effective amount of a bacterium selected from a lacticacid bacterium, a Bifidobacterium or a mixture of any thereof.

In a still further aspect, the invention provides a method of regulatinglipid absorption in a mammal in need thereof, the method comprisingadministering an effective amount of a bacterium selected from a lacticacid bacterium, a Bifidobacterium or a mixture of any thereof.

In a yet further aspect, the invention provides a method of treatingendotoxemia in a mammal in need thereof by regulating lipid absorptionin said mammal, the method comprising administering an effective amountof a bacterium selected from a lactic acid bacterium, a Bifidobacteriumor a mixture of any thereof.

In a still further aspect, the invention provides a method of treatingbacteremia in a mammal in need thereof, the method comprisingadministering an effective amount of a bacterium selected from a lacticacid bacterium, a Bifidobacterium or a mixture of any thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the level of plasma LPS (endotoxemia) in miceadministered with normal chow (NC), high fat diet (HFD), or high fatdiet supplemented with bacteria according to the present invention,namely Lactobacillus acidophilus strain NCFM (NCFM), Bifidobacteriumanimalis subsp. lactis strain 420 (B420) or a combination of the two(NCFM+B420);

FIG. 2 illustrates the average tissue level of bacteria of the familyEnterobacteriaceae (levels above detection limit) in mice administeredwith normal chow, high fat diet, or high fat diet supplemented withbacteria according to the present invention (NCFM, B420 or NCFM+B420);

FIG. 3 illustrates the average tissue level of bacteria of the genusEnterococcus (levels above detection limit) in mice administered withnormal chow, high fat diet, or high fat diet supplemented with bacteriaaccording to the present invention (NCFM, B420 or NCFM+B420);

FIG. 4 illustrates the average tissue level of bacteria of the genusLactobacillus(levels above detection limit) in mice administered withnormal chow, high fat diet, or high fat diet supplemented with bacteriaaccording to the present invention (NCFM, B420 or NCFM+B420);

FIG. 5 illustrates the average tissue level of bacteria of the classBacteroidetes (levels above detection limit) in mice administered withnormal chow, high fat diet, or high fat diet supplemented with bacteriaaccording to the present invention (NCFM, B420 or NCFM+B420);

FIG. 6 illustrates the average tissue level of Total Domain Bacterium(levels above detection limit) in mice administered with normal chow,high fat diet, or high fat diet supplemented with bacteria according tothe present invention (NCFM, 8420 or NCFM+B420);

FIG. 7 illustrates the levels of E. coli in the mucosa or differentsegments of the lumen of mice administered a high fat diet and treatedwith vehicle or a high fat diet supplemented with bacteria according tothe present invention (B420);

FIG. 8 illustrates the effect on the adhesion of Escherichia coli in themucosa of the caecum of mice administered a high fat diet and treatedwith vehicle or a high fat diet supplemented with bacteria according tothe present invention (B420), alone or in combination with a prebiotic;

FIG. 9 illustrates the effect on the translocation of Escherichia coliinto the host tissues of mice administered a high fat diet and treatedwith vehicle or a high fat diet supplemented with bacteria according tothe present invention (B420), alone or in combination with a prebiotic;and

FIG. 10 illustrates the effect on fasted insulin levels and insulinsecretion at fed state of mice administered a high fat diet and treatedwith vehicle or a high fat diet supplemented with bacteria according tothe present invention (B420), alone or in combination with a prebiotic.

DETAILED DESCRIPTION OF THE INVENTION

Lactic Acid Bacteria and Bifidobacteria

The bacterium used in embodiments of the present invention is selectedfrom a lactic acid bacterium (LAB), a Bifidobacterium or a mixture ofany thereof. In this specification the term ‘lactic acid bacterium’includes any bacterium capable of producing, as the major metabolic endproduct of carbohydrate fermentation, lactic acid or at least one of itsderivatives (including, but not limited to, acetic acid or propionicacid): the term is therefore intended to include propionic acid bacteria(PAB), which produce propionic acid as a carbohydrate fermentationproduct.

The bacterium may be used in any form capable of exerting the effectsdescribed herein. For example, the bacteria may be viable, dormant,inactivated or dead bacteria. Preferably, the bacteria are viablebacteria.

The bacteria may comprise whole bacteria or may comprise bacterialcomponents. Examples of such components include bacterial cell wallcomponents such as peptidoglycan, bacterial nucleic acids such as DNAand RNA, bacterial membrane components, and bacterial structuralcomponents such as proteins, carbohydrates, lipids and combinations ofthese such as lipoproteins, glycolipids and glycoproteins.

The bacteria may also or alternatively comprise bacterial metabolites.In this specification the term ‘bacterial metabolites’ includes allmolecules produced or modified by the (probiotic) bacteria as a resultof bacterial metabolism during growth, survival, persistence, transit orexistence of bacteria during probiotic product manufacture and storageand during gastrointestinal transit in a mammal. Examples include allorganic acids, inorganic acids, bases, proteins and peptides, enzymesand co-enzymes, amino acids and nucleic acids, carbohydrates, lipids,glycoproteins, lipoproteins, glycolipids, vitamins, all bioactivecompounds, metabolites containing an inorganic component, and all smallmolecules, for example nitrous molecules or molecules containing asulphurous acid.

Preferably the bacteria comprise whole bacteria, more preferably wholeviable bacteria.

Preferably the lactic acid bacterium and/or Bifidobacterium to be usedin the present invention is a lactic acid bacterium and/orBifidobacterium which is generally recognised as safe and, which ispreferably GRAS approved.

A skilled person will readily be aware of specific species and orstrains of lactic acid bacteria and/or Bifidobacteria from within thegenera described herein which are used in the food and/or agriculturalindustries and which are generally considered suitable for human and/oranimal consumption.

Preferably, the lactic acid bacterium and/or Bifidobacterium used inaccordance with the present invention is one which is suitable for humanand/or animal consumption.

In the present invention, the bacteria used may be of the same type(genus, species and strain) or may comprise a mixture of genera, speciesand/or strains.

Suitable lactic acid bacteria may be selected from the generaLactococcus, Lactobacillus, Leuconostoc, Carnobacterium, Enterococcus,Propionibacterium, Pediococcus, and Streptococcus and mixtures thereof.Typically, the lactic acid bacteria are selected from the speciesLeuconostoc spp., Lactococcus cremoris, Lactococcus lactis,Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefiri,Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus helveticus,Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillussalivarius, Lactobacillus curvatus, Lactobacillus bulgaricus,Lactobacillus sakei, Lactobacillus reuteri, Lactobacillus fermentum,Lactobacillus farciminis, Lactobacillus lactis, Lactobacillusdelbreuckii, Lactobacillus plantarum, Lactobacillus paraplantarum,Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsoniiand Lactobacillus jensenii, and combinations of any thereof.

Suitable Bifidobacteria are selected from the species Bifidobacteriumlactis, Bifidobacterium bifidium, Bifidobacterium longum,Bifidobacterium animalis, Bifidobacterium breve, Bifidobacteriuminfantis, Bifidobacterium catenulatum, Bifidobacteriumpseudocatenulatum, Bifidobacterium adolescentis, and Bifidobacteriumangulatum, and combinations of any thereof.

Preferably, the bacteria used in the present invention are selected fromthe genera Lactobacillus or Bifidobacterium and mixtures thereof. Morepreferably, the bacteria used in the present invention are selected fromthe species Lactobacillus acidophilus, Lactobacillus plantarum,Lactobacillus salivarius, Bifidobacterium animalis, Bifidobacteriumlactis, or Bifidobacterium bifidium, and mixtures thereof. A combinationof bacteria of the species Lactobacillus acidophilus and bacteria of thespecies Bifidobacterium animalis is especially preferred.

In a particularly preferred embodiment, the bacteria used in the presentinvention are Lactobacillus acidophilus strain NCFM. Lactobacillusacidophilus NCFM is commercially available from Danisco A/S under thename HOWARU™ Dophilus.

In an alternative particularly preferred embodiment, the bacteria usedin the present invention are Bifidobacterium animalis subsp. lactisstrain 420 (B420), This strain is commercially available from DaniscoA/S.

In an alternative particularly preferred embodiment, the bacteria usedin the present invention are Lactobacillus salivarius strain 33 (Ls-33).This strain is commercially available from Danisco A/S.

In one embodiment, the bacterium used in the present invention is aprobiotic bacterium. In this specification the term ‘probioticbacterium’ is defined as covering any non-pathogenic bacterium which,when administered live in adequate amounts, confer a health benefit onthe host. These probiotic strains generally have the ability to survivethe passage through the upper part of the digestive tract. They arenon-pathogenic, non-toxic and exercise their beneficial effect on healthon the one hand via ecological interactions with the resident flora inthe digestive tract, and on the other hand via their ability toinfluence the immune system in a positive manner via the “GALT”(gut-associated lymphoid tissue). Depending on the definition ofprobiotics, these bacteria, when given in a sufficient number, have theability to progress live through the intestine, however they do notcross the intestinal barrier and their primary effects are thereforeinduced in the lumen and/or the wall of the gastrointestinal tract. Theythen form part of the resident flora during the administration period.This colonization (or transient colonization) allows the probioticbacteria to exercise a beneficial effect, such as the repression ofpotentially pathogenic micro-organisms present in the flora andinteractions with the immune system of the intestine.

In preferred embodiments, the bacterium used in the present invention isa probiotic lactic acid bacterium and/or a probiotic Bifidobacterium.

in some preferred embodiments, the Bifidobacterium is used in thepresent invention together with a bacterium of the genus Lactobacillus.A combination of Bifidobacterium and Lactobacillus bacteria according tothe present invention exhibits a synergistic effect in certainapplications (i.e. an effect which is greater than the additive effectof the bacteria when used separately). For example, combinations which,in addition to having effect on the mammal as single components, mayhave beneficial effect on the other components of the combination, forexample by producing metabolites which are then in turn used as anenergy source by other components of the combination, or maintainingphysiological conditions which favour the other components.

Typically, the Lactobacillus bacteria used in the combination areselected from the species Lactobacillus acidophilus, Lactobacilluscasei, Lactobacillus kefiri, Lactobacillus bifidus, Lactobacillusbrevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillusrhamnosus, Lactobacillus salivarius, Lactobacillus curvatus,Lactobacillus bulgaricus, Lactobacillus sakei, Lactobacillus reuteri,Lactobacillus fermentum, Lactobacillus farciminis, Lactobacillus lactis,Lactobacillus delbreuckii, Lactobacillus plantarum, Lactobacillusparaplantarum, Lactobacillus crispatus, Lactobacillus gasseri,Lactobacillus johnsonii and Lactobacillus jensenii, and combinations ofany thereof.

In preferred embodiments, the Lactobacillus bacterium used in thepresent invention is a probiotic Lactobacillus.

Preferably, the Lactobacillus bacterium used in the present invention ofthe species Lactobacillus acidophilus.

In a particularly preferred embodiment, the bacteria used in the presentinvention comprise a combination of Bifidobacterium animalis subsp.lactis strain 420 (B420) and Lactobacillus acidophilus strain NCFM.

Dosage

The lactic acid bacterium and/or Bifidobacterium used in accordance withthe present invention (such as a strain of Lactobacillus spp.; forexample a strain of Lactobacillus acidophilus, Lactobacillus salivariusand/or Lactobacillus plantarum, such as a strain of Lactobacillusacidophilus or Lactobacillus salivarius, for example Lactobacillusacidophilus strain NCFM or Lactobacillus salivarius strain 33) and/or astrain of Bifidobacterium spp., such as a strain of Bifidobacteriumanimalis subsp. lactis, for example Bifidobacterium animalis subsp.lactis strain 420 (B420)), may comprise from 10⁶ to 10¹² CFU ofbacteria/g of support, and more particularly from 10⁸ to 10¹² CFU ofbacteria/g of support, preferably 10⁹ to 10¹² CFU/g for the lyophilizedform.

Suitably the lactic acid bacterium and/or Bifidobacterium used inaccordance with the present invention (such as a strain of Lactobacillusspp.; for example a strain of Lactobacillus acidophilus, Lactobacillussalivarius and/or Lactobacillus plantarum, such as a strain ofLactobacillus acidophilus or Lactobacillus salivarius, for exampleLactobacillus acidophilus strain NCFM or Lactobacillus salivarius strain33) and/or a strain of Bifidobacterium spp., such as a strain ofBifidobacterium animalis subsp. lactis, for example Bifidobacteriumanimalis subsp. lactis strain 420 (B420)), may be administered at adosage of from about 10⁶ to about 10¹² CFU of microorganism/dose,preferably about 10⁸ to about 10¹² CFU of microorganism/dose. By theterm “per dose” it is meant that this amount of microorganism isprovided to a subject either per day or per intake, preferably per day.For example, if the microorganism is to be administered in a foodproduct (for example in a yoghurt)—then the yoghurt will preferablycontain from about 10⁸ to 10¹² CFU of the microorganism. Alternatively,however, this amount of microorganism may be split into multipleadministrations each consisting of a smaller amount of microbialloading—so long as the overall amount of microorganism received by thesubject in any specific time (for instance each 24 hour period) is fromabout 10⁶ to about 10¹² CFU of microorganism, preferably 10⁸ to about10¹² CFU of microorganism.

In accordance with the present invention an effective amount of at leastone strain of a microorganism may be at least 10⁶ CFU ofmicroorganism/dose, preferably from about 10⁶ to about 10¹² CFU ofmicroorganism/dose, preferably about 10⁸ to about 10¹² CFU ofmicroorganism/dose.

In one embodiment, preferably the lactic acid bacterium and/orBifidobacterium used in accordance with the present invention (such as astrain of Lactobacillus spp.; for example a strain of Lactobacillusacidophilus, Lactobacillus salivarius and/or Lactobacillus plantarumand/or a strain of Bifidobacterium spp., such as a strain ofLactobacillus acidophilus or Lactobacillus salivarius, for exampleLactobacillus acidophilus strain NCFM or Lactobacillus salivarius strain33) such as a strain of Bifidobacterium animalis subsp. lactis, forexample Bifidobacterium animalis subsp. lactis strain 420 (B420)) may beadministered at a dosage of from about 10⁶ to about 10¹² CFU ofmicroorganism/day, preferably about 10⁸ to about 10¹² CFU ofmicroorganism/day. Hence, the effective amount in this embodiment may befrom about 10⁶ to about 10¹² CFU of microorganism/day, preferably about10⁸ to about 10¹² CFU of microorganism/day.

CFU stands for “colony-forming units”. By ‘support’ is meant the foodproduct, dietary supplement or the pharmaceutically acceptable support.

Subjects/Medical Indications

The lactic acid bacteria and/or Bifidobacteria to which the presentinvention relates are administered to a mammal, including for examplelivestock (including cattle, horses, pigs, chickens and sheep), andhumans. In some aspects of the present invention the mammal is acompanion animal (including pets), such as a dog or a cat for instance.In some aspects of the present invention, the subject may suitably be ahuman.

The inventors have surprisingly found that the lactic acid bacteriaand/or Bifidobacteria to which the present invention relates are capableof lowering blood plasma lipopolysaccharides (LPS) levels. This findingconfers the potential for the bacteria to be useful in the treatment ofendotoxemia, in particular metabolic endotoxemia.

LPS is a major cell wall component of gram-negative bacteria, whichoccurs in the intestine as a component of gram-negative microbiota butalso as free LPS. Reduction of metabolic endotoxemia may result fromreduction of translocation of gram-negative bacteria into the host,reduction of absorption of free LPS by the host, or enhanced clearanceof LPS by the host.

In this specification, the term ‘endotoxemia’ when used alone means thepresence of an elevated level of lipopolysaccharides (also known asendotoxins) in the body (particularly, although not exclusively, inblood plasma) when compared with basal lipopolysaccharide levels. Theterm ‘endotoxemia’ when used alone is intended to encompass bothmetabolic endotoxemia, defined in more detail below, and endotoxemia ofother etiologies, such as endotoxemia caused by pathogenic infections(especially bacterial infections) or endotoxemia caused bysmall-intestinal bacterial overgrowth (SIBO).

In some embodiments, the lactic acid bacteria and/or Bifidobacteria towhich the present invention relates are used to treat metabolicendotoxemia. In one aspect, the term ‘metabolic endotoxemia’ meansendotoxemia (as defined above) induced by a high-fat diet. The term‘high-fat diet’ is defined in more detail below.

In one aspect, the term ‘metabolic endotoxemia’ means an increase of thelevel of lipopolysaccharides in the mammalian body (particularly,although not exclusively, in blood plasma) by a factor ranging from 1.5to 20, preferably 2 to 10, such as 2 to 4, preferably 2 to 3.5, comparedwith basal (normal) mammalian lipopolysaccharide levels. The increase inlipopolysaccharide levels is typically measured by the Limulusamaebocyte assay, a test well known to those skilled in the art.

In contrast, in the case of endotoxemia caused by pathogenic infections,such as bacterial infections (septic shock endotoxemia), LPS levels inthe mammalian body, especially the human body (particularly, althoughnot exclusively, in blood) are typically raised by a factor of greaterthan 20, such as greater than 30, preferably greater than 50, such asgreater than 70, such as greater than 100, such as greater than 150,such as greater than 200 times compared with basal (normal) mammalianlipopolysaccharide levels.

Basal levels of lipopolysaccharides in humans are typically in the rangeof 1-2 Endotoxin Units (EU) per ml, as measured by the Limulusamaebocyte assay, an example of which is the Limulus amaebocyte extractassay with Kinetic-QCL test (Bio Whittaker, Cambrex BioScience).

Therefore in an alternative aspect, the term ‘metabolic endotoxemia’means a level of lipopolysaccharides in the body (particularly, althoughnot exclusively, in blood plasma) ranging from 1.5 to 40, preferably 2to 20, such as 2 to 8, preferably 2 to 7, Endotoxin Units (EU)/ml, asmeasured by the Limulus amaebocyte extract assay.

In contrast, in the case of endotoxemia caused by pathogenic infections,such as bacterial infections (septic shock endotoxemia), LPS levels inthe mammalian body, especially the human body (particularly, althoughnot exclusively, in blood) are typically greater than 40, such asgreater than 60, preferably greater than 100, such as greater than 140,such as greater than 200, such as greater than 300, such as greater than400 EU/ml, as measured by the Limulus amaebocyte extract assay.

The present inventors have also surprisingly found that the lactic acidbacteria and/or Bifidobacteria to which the present invention relatescan reduce the level of bacteria in metabolically very importanttissues, the mesenteric adipose tissue, subcutaneous adipose tissue,mesenteric ganglion and subcutaneous ganglion, and the liver and spleen.This confers the potential for lactic acid bacteria and/orBifidobacteria to which the present invention relates to be used forpreventing or treating bacterial translocation into tissues andpreventing or treating metabolic endotoxemia.

In particularly preferred embodiments, the lactic acid bacteria and/orBifidobacteria to which the present invention relates may be used toreduce the levels of bacteria of the family Enterobacteriaceae in themesenteric adipose tissue and the class Bacteriodetes in the livertissue. These two major bacterial groups contain LPS as a component ofbacterial cell wall and therefore act as potential source of elevatedplasma LPS, which is associated with metabolic endotoxemia. Thesetissues play particularly important role in metabolic endotoxemia andother metabolic diseases, since LPS causes inflammation in thesetissues, potentially leading to a cascade of adverse events includingimpaired glucose metabolism and reduced insulin sensitivity.

The present inventors have also surprisingly found that the lactic acidbacteria and/or Bifidobacteria to which the present invention relatesare capable of regulating lipid absorption. Without wishing to be boundby theory, it is believed that, as LPS are carried primarily in lipids,regulating lipid absorption reduces the amount of bacteria and thereforethe amount of LPS passing the intestinal barrier. This confers thepotential for lactic acid bacteria and/or Bifidobacteria to which thepresent invention relates to be used for preventing the passage of LPSinto tissues and therefore preventing or treating metabolic endotoxemia.

In addition, the present inventors have surprisingly found that thelactic acid bacteria and/or Bifidobacteria to which the presentinvention relates are capable of reducing elevated gram-negativebacterial adhesion in the gastrointestinal tract. Without wishing to bebound by theory, it is believed that reducing the elevated gram-negativebacterial adhesion reduces the amount of bacteria and therefore theamount of LPS passing the intestinal barrier. This confers the potentialfor lactic acid bacteria and/or Bifidobacteria to which the presentinvention relates to be used for preventing the passage of LPS intotissues and therefore preventing or treating metabolic endotoxemia.

The lactic acid bacteria and/or Bifidobacteria to which the presentinvention relates are suitable for administration to both diabetic andobese mammals. They could also be suitable for diabetic and non-obesemammals, as well as to obese mammals possessing the risk factors fordiabetes, but not yet in a diabetic state. This aspect is discussed inmore detail below.

In preferred embodiments, the condition being treated or prevented isdiet-induced and/or diet-associated. The present inventors havesurprisingly found that the lactic acid bacteria and/or Bifidobacteriacan be used in accordance with the present invention to treat a numberof diet-induced and/or diet-associated conditions, as described in moredetail herein.

In particular, the use of lactic acid bacteria and/or Bifidobacteriaaccording to the present invention is suitable for the treatment ofmammals ingesting a high-fat diet. This aspect is discussed in moredetail below.

The compositions are suitable for use in obese and diabetic patients. Inthis specification the term ‘diabetes’ includes all forms of diabeteswhich, as noted above, is characterised by disordered metabolism andabnormally high blood sugar (hyperglycaemia) resulting from insufficientlevels of the hormone insulin. The term therefore includes Type 1diabetes, Type 2 diabetes, gestational diabetes, and impaired glucosetolerance. Type 1 diabetes is characterised by loss of theinsulin-producing beta cells of the islets of Langerhans in thepancreas, leading to a deficiency of insulin. Type 2 diabetes mellitusis characterised by insulin resistance or reduced insulin sensitivity,combined with reduced insulin secretion. Gestational diabetes isformally defined as “any degree of glucose intolerance with onset orfirst recognition during pregnancy”. Impaired Glucose Tolerance (IGT) isa pre-diabetic state of dysglycemia that is associated with insulinresistance and increased risk of cardiovascular pathology. According tothe criteria of the World Health Organization and the American DiabetesAssociation, impaired glucose tolerance is defined as two-hour glucoselevels of 140 to 199 mg per dL (7.8 to 11.0 mmol) on the 75-g oralglucose tolerance test. A patient is said to be under the condition ofIGT when he/she has an intermediately raised glucose level after 2hours, but less than would qualify for type 2 diabetes mellitus. Thefasting glucose may be either normal or mildly elevated. IGT may precedetype 2 diabetes mellitus by many years. IGT is also a risk factor formortality.

In this specification, the term obesity is linked to body mass index(BMI). The body mass index (BMI) (calculated as weight in kilogramsdivided by the square of height in metres) is the most commonly acceptedmeasurement for overweight and/or obesity. A BMI exceeding 25 isconsidered overweight. Obesity is defined as a BMI of 30 or more, with aBMI of 35 or more considered as serious comorbidity obesity and a BMI of40 or more considered morbid obesity.

As noted above, the term “obesity” as used herein includes obesity,comorbidity obesity and morbid obesity. Therefore, the term “obese” asused here may be defined as a subject having a BMI of more than or equalto 30. In some embodiments, suitably an obese subject may have a BMI ofmore than or equal to 30, suitably 35, suitably 40.

While the composition of the invention is particularly suitable for usein patients who are both diabetic and obese, the composition is alsosuitable for those who are diabetic but not obese. It may also besuitable for use in obese patients possessing the risk factors fordiabetes, but not yet in a diabetic state, as it could be expected thatan obese person (but not diabetic), could limit the metabolicconsequences of his obesity, i.e. the diabetes or at leastinsulino-resistance development.

In this specification the term “treatment” or “treating” refers to anyadministration of the lactic acid bacteria and/or Bifidobacteriaaccording to the present invention and includes: (1) preventing thespecified disease from occurring in an animal which may be predisposedto the disease but does not yet experience or display the pathology orsymptomatology of the disease (including prevention of one or more riskfactors associated with the disease); (2) inhibiting the disease in ananimal that is experiencing or displaying the pathology orsymptomatology of the diseased (i.e., arresting further development ofthe pathology and/or symptomatology), or (3) ameliorating the disease inan animal that is experiencing or displaying the pathology orsymptomatology of the diseased (i.e., reversing the pathology and/orsymptomatology).

Diet

As noted above, diabetic and/or obese mammals treated with bacteriaaccording to the present invention may continue to ingest a high-fatdiet while mitigating the metabolic consequences of their condition(s).In this specification the term ‘high-fat diet’ means a diet generallycontaining at least 20%, preferably at least 25%, such as at least 30%,for example at least 35%, such as at least 40%, for example at least45%, such as at least 50%, for example at least 55%, such as at least60%, for example at least 65%, such as at least 70%, for example atleast 75%, such as at least 80%, for example at least 85%, such as atleast 90% of calories from fat.

In some embodiments, diabetic and/or obese mammals treated with bacteriaaccording to the present invention may ingest a high-carbohydrate dietwhile mitigating the metabolic consequences of their condition(s). Inthis specification the term ‘high-carbohydrate diet’ means a dietgenerally containing at least 50%, for example at least 55%, such as atleast 60%, for example at least 65%, such as at least 70%, for exampleat least 75%, such as at least 80%, for example at least 85%, such as atleast 90% of calories from carbohydrate.

Compositions

While is it possible to administer lactic acid bacteria and/orBifidobacteria alone according to the present invention, the lactic acidbacteria and/or Bifidobacteria are typically and preferably administeredon or in a support as part of a product, in particular as a component ofa food product, a dietary supplement or a pharmaceutical formulation.These products typically contain additional components well known tothose skilled in the art.

Any product which can benefit from the composition may be used in thepresent invention. These include but are not limited to foods,particularly fruit conserves and dairy foods and dairy food-derivedproducts, and pharmaceutical products. The lactic acid bacteria may bereferred to herein as “the composition of the present invention” or “thecomposition”.

Food

In one embodiment, the lactic acid bacteria and/or Bifidobacteria areemployed according to the invention in a food product such as a foodsupplement, a drink or a powder based on milk. Here, the term “food” isused in a broad sense—and covers food for humans as well as food foranimals (i.e. a feed). In a preferred aspect, the food is for humanconsumption.

The food may be in the form of a solution or as a solid—depending on theuse and/or the mode of application and/or the mode of administration.

When used as, or in the preparation of, a food, such as functional food,the composition of the present invention may be used in conjunction withone or more of: a nutritionally acceptable carrier, a nutritionallyacceptable diluent, a nutritionally acceptable excipient, anutritionally acceptable adjuvant, a nutritionally active ingredient.

By way of example, the composition of the present invention can be usedas an ingredient to soft drinks, a fruit juice or a beverage comprisingwhey protein, health teas, cocoa drinks, milk drinks and lactic acidbacteria drinks, yoghurt and drinking yoghurt, cheese, ice cream, waterices and desserts, confectionery, biscuits cakes and cake mixes, snackfoods, balanced foods and drinks, fruit fillings, care glaze, chocolatebakery filling, cheese cake flavoured filling, fruit flavoured cakefilling, cake and doughnut icing, instant bakery filling creams,fillings for cookies, ready-to-use bakery filling, reduced caloriefilling, adult nutritional beverage, acidified soy/juice beverage,aseptic/retorted chocolate drink, bar mixes, beverage powders, calciumfortified soy/plain and chocolate milk, calcium fortified coffeebeverage.

The composition can further be used as an ingredient in food productssuch as American cheese sauce, anti-caking agent for grated & shreddedcheese, chip dip, cream cheese, dry blended whip topping fat free sourcream, freeze/thaw dairy whipping cream, freeze/thaw stable whippedtipping, low fat and light natural cheddar cheese, low fat Swiss styleyoghurt, aerated frozen desserts, hard pack ice cream, label friendly,improved economics & indulgence of hard pack ice cream, low fat icecream: soft serve, barbecue sauce, cheese dip sauce, cottage cheesedressing, dry mix Alfredo sauce, mix cheese sauce, dry mix tomato sauceand others.

The term “dairy product” as used herein is meant to include a mediumcomprising milk of animal and/or vegetable origin. As milk of animalorigin there can be mentioned cow's, sheep's, goat's or buffalo's milk.As milk of vegetable origin there can be mentioned any fermentablesubstance of vegetable origin which can be used according to theinvention, in particular originating from soybeans, rice or cereals.

Still more preferably the food product employed according to theinvention is a fermented milk or humanized milk.

For certain aspects, preferably the present invention may be used inconnection with yoghurt production, such as fermented yoghurt drink,yoghurt, drinking yoghurt, cheese, fermented cream, milk based dessertsand others.

Suitably, the composition can be further used as an ingredient in one ormore of cheese applications, meat applications, or applicationscomprising protective cultures.

The present invention also provides a method of preparing a food or afood ingredient, the method comprising admixing the compositionaccording to the present invention with another food ingredient.

Advantageously, the present invention relates to products that have beencontacted with the composition of the present invention (and optionallywith other components/ingredients), wherein the composition is used inan amount to be capable of improving the nutrition and/or healthbenefits of the product.

As used herein the term “contacted” refers to the indirect or directapplication of the composition of the present invention to the product.Examples of the application methods which may be used, include, but arenot limited to, treating the product in a material comprising thecomposition, direct application by mixing the composition with theproduct, spraying the composition onto the product surface or dippingthe product into a preparation of the composition.

Where the product of the invention is a foodstuff, the composition ofthe present invention is preferably admixed with the product.Alternatively, the composition may be included in the emulsion or rawingredients of a foodstuff. In a further alternative, the compositionmay be applied as a seasoning, glaze, colorant mixture, and the like.

For some applications, it is important that the composition is madeavailable on or to the surface of a product to be affected/treated. Thisallows the composition to impart one or more of the following favourablecharacteristics: nutrition and/or health benefits.

The compositions of the present invention may be applied to intersperse,coat and/or impregnate a product with a controlled amount of a viablemicroorganism.

Preferably, the composition is used to ferment milk or sucrose fortifiedmilk or lactic media with sucrose and/or maltose where the resultingmedia containing all components of the composition—i.e. saidmicroorganism according to the present invention—can be added as aningredient to yoghurt milk in suitable concentrations—such as forexample in concentrations in the final product which offer a daily doseof 10⁶-10¹⁰ cfu. The microorganism according to the present inventionmay be used before or after fermentation of the yoghurt.

For some aspects the microorganisms according to the present inventionare used as, or in the preparation of, animal feeds, such as livestockfeeds, in particular poultry (such as chicken) feed, or pet food.

Advantageously, where the product is a food product, the lactic acidbacteria should remain effective through the normal “sell-by” or“expiration” date during which the food product is offered for sale bythe retailer. Preferably, the effective time should extend past suchdates until the end of the normal freshness period when food spoilagebecomes apparent. The desired lengths of time and normal shelf life willvary from foodstuff to foodstuff and those of ordinary skill in the artwill recognise that shelf-life times will vary upon the type offoodstuff, the size of the foodstuff, storage temperatures, processingconditions, packaging material and packaging equipment.

Food Ingredient

The composition of the present invention may be used as a foodingredient and/or feed ingredient.

As used herein the term “food ingredient” or “feed ingredient” includesa formulation which is or can be added to functional foods or foodstuffsas a nutritional supplement.

The food ingredient may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

Food Supplements

The composition of the present invention may be—or may be added to—foodsupplements (also referred to herein as dietary supplements).

Functional Foods

The composition of the present invention may be—or may be addedto—functional foods.

As used herein, the term “functional food” means food which is capableof providing not only a nutritional effect, but is also capable ofdelivering a further beneficial effect to consumer.

Accordingly, functional foods are ordinary foods that have components oringredients (such as those described herein) incorporated into them thatimpart to the food a specific functional—e.g. medical or physiologicalbenefit—other than a purely nutritional effect.

Although there is no legal definition of a functional food, most of theparties with an interest in this area agree that they are foods marketedas having specific health effects beyond basic nutritional effects.

Some functional foods are nutraceuticals. Here, the term “nutraceutical”means a food which is capable of providing not only a nutritional effectand/or a taste satisfaction, but is also capable of delivering atherapeutic (or other beneficial) effect to the consumer. Nutraceuticalscross the traditional dividing lines between foods and medicine.

Medicament

The term “medicament” as used herein encompasses medicaments for bothhuman and animal usage in human and veterinary medicine. In addition,the term “medicament” as used herein means any substance which providesa therapeutic and/or beneficial effect. The term “medicament” as usedherein is not necessarily limited to substances which need MarketingApproval, but may include substances which can be used in cosmetics,nutraceuticals, food (including feeds and beverages for example),probiotic cultures, and natural remedies. In addition, the term“medicament” as used herein encompasses a product designed forincorporation in animal feed, for example livestock feed and/or petfood.

Pharmaceutical

The composition of the present invention may be used as—or in thepreparation of a pharmaceutical. Here, the term “pharmaceutical” is usedin a broad sense—and covers pharmaceuticals for humans as well aspharmaceuticals for animals (i.e. veterinary applications). In apreferred aspect, the pharmaceutical is for human use and/or for animalhusbandry.

The pharmaceutical can be for therapeutic purposes—which may be curativeor palliative or preventative in nature. The pharmaceutical may even befor diagnostic purposes.

A pharmaceutically acceptable support may be for example a support inthe form of compressed tablets, tablets, capsules, ointments,suppositories or drinkable solutions. Other suitable forms are providedbelow.

When used as—or in the preparation of—a pharmaceutical, the compositionof the present invention may be used in conjunction with one or more of:a pharmaceutically acceptable carrier, a pharmaceutically acceptablediluent, a pharmaceutically acceptable excipient, a pharmaceuticallyacceptable adjuvant, a pharmaceutically active ingredient.

The pharmaceutical may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

The lactic acid bacteria and/or Bifidobacteria to which the presentinvention relates may be used as pharmaceutical ingredients. Here, thecomposition may be the sole active component or it may be at least oneof a number (i.e. 2 or more) of active components.

The pharmaceutical ingredient may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

The lactic acid bacteria and/or Bifidobacteria to which the presentinvention relates may be used in any suitable form—whether when alone orwhen present in a combination with other components or ingredients. Thelactic acid bacteria and/or Bifidobacteria to which the presentinvention relates may be referred to herein as “the composition”.Likewise, combinations comprising the composition of the presentinvention and other components and/or ingredients (i.e. ingredients—suchas food ingredients, functional food ingredients or pharmaceuticalingredients) may be used in any suitable form.

The lactic acid bacteria and/or Bifidobacteria to which the presentinvention relates may be used in the form of solid or liquidpreparations or alternatives thereof. Examples of solid preparationsinclude, but are not limited to tablets, capsules, dusts, granules andpowders which may be wettable, spray-dried or freeze-dried. Examples ofliquid preparations include, but are not limited to, aqueous, organic oraqueous-organic solutions, suspensions and emulsions.

Suitable examples of forms include one or more of: tablets, pills,capsules, ovules, solutions or suspensions, which may contain flavouringor colouring agents, for immediate-, delayed-, modified-, sustained-,pulsed- or controlled-release applications.

By way of example, if the composition of the present invention is usedin a tablet form—such for use as a functional ingredient—the tablets mayalso contain one or more of: excipients such as microcrystallinecellulose, lactose, sodium citrate, calcium carbonate, dibasic calciumphosphate and glycine; disintegrants such as starch (preferably corn,potato or tapioca starch), sodium starch glycollate, croscarmellosesodium and certain complex silicates; granulation binders such aspolyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),hydroxypropylcellulose (HPC), sucrose, gelatin and acacia; lubricatingagents such as magnesium stearate, stearic acid, glyceryl behenate andtalc may be included.

Examples of nutritionally acceptable carriers for use in preparing theforms include, for example, water, salt solutions, alcohol, silicone,waxes, petroleum jelly, vegetable oils, polyethylene glycols, propyleneglycol, liposomes, sugars, gelatin, lactose, amylose, magnesiumstearate, talc, surfactants, silicic acid, viscous paraffin, perfumeoil, fatty acid monoglycerides and diglycerides, petroethral fatty acidesters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

Preferred excipients for the forms include lactose, starch, a cellulose,milk sugar or high molecular weight polyethylene glycols.

For aqueous suspensions and/or elixirs, the composition of the presentinvention may be combined with various sweetening or flavouring agents,colouring matter or dyes, with emulsifying and/or suspending agents andwith diluents such as water, propylene glycol and glycerin, andcombinations thereof.

The forms may also include gelatin capsules; fibre capsules, fibretablets etc.; or even fibre beverages.

Further examples of form include creams. For some aspects themicroorganism used in the present invention may be used inpharmaceutical and/or cosmetic creams such as sun creams and/orafter-sun creams for example.

In one aspect, the composition according to the present invention may beadministered in an aerosol, for example by way of a nasal spray, forinstance for administration to the respiratory tract.

Prebiotics

The composition of the present invention may additionally contain one ormore prebiotics. Prebiotics are a category of functional food, definedas non-digestible food ingredients that beneficially affect the host byselectively stimulating the growth and/or activity of one or a limitednumber of bacteria (particularly, although not exclusively, probiotics,Bifidobacteria and/or lactic acid bacteria) in the colon, and thusimprove host health. Typically, prebiotics are carbohydrates (such asoligosaccharides), but the definition does not precludenon-carbohydrates. The most prevalent forms of prebiotics arenutritionally classed as soluble fibre. To some extent, many forms ofdietary fibre exhibit some level of prebiotic effect.

In one embodiment, a prebiotic is a selectively fermented ingredientthat allows specific changes, both in the composition and/or activity inthe gastrointestinal microflora that confers benefits upon hostwell-being and health.

Suitably, the prebiotic may be used according to the present inventionin an amount of 0.01 to 100 g/day, preferably 0.1 to 50 g/day, morepreferably 0.5 to 20 g/day. In one embodiment, the prebiotic may be usedaccording to the present invention in an amount of 1 to 100 g/day,preferably 2 to 9 g/day, more preferably 3 to 8 g/day. In anotherembodiment, the prebiotic may be used according to the present inventionin an amount of 5 to 50 g/day, preferably 10 to 25 g/day.

Examples of dietary sources of prebiotics include soybeans, inulinsources (such as Jerusalem artichoke, jicama, and chicory root), rawoats, unrefined wheat, unrefined barley and yacon.

Examples of suitable prebiotics include alginate, xanthan, pectin,locust bean gum (LBG), inulin, guar gum, galacto-oligosaccharide (GOS),fructo-oligosaccharide (FOS), polydextrose (i.e. Litesse®), lactitol,lactosucrose, soybean oligosaccharides, isomaltulose (Palatinose™),isomalto-oligosaccharides, gluco-oligosaccharides,xylo-oligosaccharides, manno-oligosaccharides, beta-glucans, cellobiose,raffinose, gentiobiose, melibiose, xylobiose, cyclodextrins, isomaltose,trehalose, stachyose, panose, pullulan, verbascose, galactomannans, andall forms of resistant starches. A particularly preferred example of aprebiotic is polydextrose.

In a yet further aspect, the invention provides use of a combination of:

-   (a) a lactic acid bacterium, a Bifidobacterium or a mixture of any    thereof; and-   (b) a prebiotic;-   in the manufacture of a food product, dietary supplement or    medicament for reducing fasted insulin levels in a mammal.

In a yet further aspect, the invention provides use of a combination of:

-   (a) a lactic acid bacterium, a Bifidobacterium or a mixture of any    thereof; and-   (b) a prebiotic;-   in the manufacture of a food product, dietary supplement or    medicament for increasing insulin secretion at fed state in a    mammal.

It is envisaged within the scope of the present invention that theembodiments of the invention can be combined such that combinations ofany of the features described herein are included within the scope ofthe present invention. In particular, it is envisaged within the scopeof the present invention that any of the therapeutic effects of thebacteria may be exhibited concomitantly.

EXAMPLES Example 1

Materials and Methods

Animal Model and Probiotic Treatment

A cohort of fifty C57BI/6 10-wk-old male mice were fed a Normal Chow(NC) (A03, SAFE, Augy, France), or a high-fat diet (HFD) (comprising 72%fat (corn oil and lard), 28% protein and <1% carbohydrates) (SAFE, Augy,France) for 4 weeks. This diet has the peculiar advantage to inducediabetes before the onset of obesity (see for example Cani et al. 2008“Role of gut microflora in the development of obesity and insulinresistance following high-fat diet feeding”. Pathol Biol (Paris); Caniet al, Diabetes, 2008, 57, 1470-81; Knauf et al. Endocrinology 2008,149, 476877; Cani et al., Diabetologia 2007, 50, 2374-83; Cani et al.Diabetes 2007, 56, 1761-1772 and Turini et al. Swiss Med Wkly 2007, 137,700-4).

Following the high fat diet feeding (before probiotic feeding), the miceunderwent an intraperitoneal glucose tolerance test. The area undercurve was calculated and the mice dispatched homogeneously according tothe different experimental groups or ten mice per group (10 mice pergroup). The data showed that all mice were in diabetic state beforeprobiotic feeding. The mice were fed four more weeks with a normal chow(n=10) or a HFD (n=40). The HFD mice were treated daily for 4 weeks withone of the following:

-   1. Vehicle treated-   2. Bifidobacterium animalis subsp. lactis strain 420 (B420)    (10⁹/bacteria per mouse)-   3. Lactobacillus acidophilus NCFM (NCFM) (10⁹/bacteria per mouse),-   4. NCFM+B420 (5×10⁸ B420+5×10⁸ NCFM per mouse).

The mice were housed in a controlled environment (inverted 12-h daylightcycle, light off at 10:00 a.m.).

Endotoxemia and Plasma CD14

Blood samples were collected from the mice in the end of the probioticor control treatments. Endotoxin assay based on a Limulus amaebocyteextract with Kinetic-QCL test (Bio Whittaker, Cambrex BioScience) wasused for the quantification of plasma LPS (endotoxemia)—this is aquantitative, kinetic assay for the detection of Gram-negative bacterialendotoxin. Sera are diluted 1/20 to 1/100 and heated during rounds of 10min at 70° C. The lower limit of detection of LPS was 0.001 EndotoxinUnits/ml. Plasma sCD14, the LPS receptor, was measured using animmuno-enzymatic method (IBL, GmbH, Hamburg, Germany).

Quantification of Microbial DNA in Mouse Tissues

Following sacrifice, the following mouse tissues were harvestedaseptically: mesenteric adipose tissue, subcutaneous adipose tissue, andliver. The tissues were snap-frozen and kept frozen until analysis.Prior to DNA extraction, tissues were homogenized using a Precellys 24automatic biological sample lyser (Bertin Technologies, Tarnos, France).Samples were weighted and diluted with 1.4 ml ASL Buffer (Qiagen,Hilden, Germany) in CK14 tubes containing ceramic beads (Bertin).Samples were milled with beads for 3×30 seconds at 6,800 rpm and kept onice in between the runs. Supernatant was collected and transferred intoVK01 tubes containing glass powder (Bertin) and the milling process wasrepeated. Supernatant was again collected and transferred into 2 mlEppendorf tubes. Bacterial DNA was isolated and purified using QIAmp DNAStool Mini Kit (Qiagen) according to the manufacturer's instructions.

Bacterial DNA was measured by quantitative real-time PCR using group orgenus specific primers. The target bacterial groups (and the primers andconditions used) were: total domain Bacteria (unpublished), familyEnterobacteriaceae (Matsuda et al. App. Env. Microbiol., 2007, 73,32-39), genus Bifidobacterium (Mäkivuokko et al. Nutrition and Cancer2005, 52(1), 93-103), genus Lactobacillus (Walter et al. Appl. Environ.Microbiol. 2001, 67, 2578-2585, Heilig et al Appl. Environ. Microbiol.2002, 68, 114-123), class Bacteroidetes and genus Enterococcus (Rinttiläet al. J. Appl. Microbiol., 2004, 97, 1166-1177). The equipment used forthe quantitative PCR analysis was Applied Biosystems 7500 FAST Real-timePCR System or ABI Prism 7000 Sequence Detection System (AppliedBiosystems, Foster City, Calif., USA). The results were expressed as logcells per gram of tissue above the detection limit of each bacterialgroup in each tissue. The detection limits were determined separatelyfor each bacterial group in each tissue type.

Results

Endotoxemia and Plasma CD14

High fat diet (HFD) was associated with 2-3 times average increase incomparison to the basal endotoxemia (basal level of plasma LPS) of themice fed with normal chow (NC) (FIG. 1). This increase in plasma LPSassociated with high fat diet is defined as metabolic endotoxemia.

All probiotic treatments were able to reduce the metabolic endotoxemiasignificantly in the mice and reverse the adverse effects of high fatdiet. The average level of plasma sCD14, the main receptor of LPS, ofmice treated with probiotics and high fat diet was over four times lowerthan mice who received high fat diet only.

Taken together, the results demonstrate that probiotic treatment reducesmetabolic endotoxemia and in addition reduces the circulating receptorsof LPS, which are also elevated with high fat diet. LPS is a major cellwall component of gram-negative bacteria, which occurs in the intestineas a component of gram-negative microbiota but also as free LPS.Reduction of metabolic endotoxemia may result from reduction oftranslocation of gram-negative bacteria into the host, reduction ofabsorption of free LPS by the host, or enhanced clearance of LPS by thehost. Probiotics can therefore be used as an innovative method forpreventing of treating metabolic endotoxemia.

Reduction of Bacterial Translocation into Tissues

High fat diet was associated with elevated levels of bacterial DNA inthe tissues, as compared to normal chow. The impact was particularlyevident in mesenteric adipose tissue, where elevated levels of thefamily Enterobacteriaceae (FIG. 2), genus Enterococcus (FIG. 3), genusLactobacillus (FIG. 4) and total Domain Bacterium (FIG. 6) wereassociated with high fat diet. In subcutaneous adipose tissue, elevatedlevels of class Bacteroidetes were detected in mice fed with high fatdiet (FIG. 5). Genus Bifidobacterium was not detected in significantamounts in any of the tissues.

Probiotic treatment significantly reduced the levels of bacterial DNA inthe tissues. In mesenteric adipose tissue, the B420 treatmentsignificantly reduced the level of family Enterobacteriaceae (FIG. 2),and the NCFM treatment showed a trend for reduced levels of familyEnterobacteriaceae (FIG. 2) and genus Lactobacillus (FIG. 4). in theliver, the combination of NCFM and B420 tended to reduce the level ofgenus Lactobacillus (FIG. 4), while NCFM treatment alone tended toreduce the levels of class Bacteroidetes (FIG. 5) and the DomainBacterium (FIG. 6), and the 8420 treatment tended to reduce the levelsof class Bacteroidetes (FIG. 5). Statistically non-significant downwardtrends were also observed for other bacteria and tissues (FIGS. 2 to 6).

The results demonstrate that probiotic treatment can reduce the level ofbacteria in metabolically very important tissues, the mesenteric adiposetissue and the liver. Thereby, probiotics may be used as an innovativemethod for preventing or treating bacterial translocation into tissues,associated with high fat diet and metabolic endotoxemia. Physiologicallyparticularly important may be the reduction of family Enterobacteriaceaein the mesenteric adipose tissue and the class Bacteriodetes in theliver tissue. These two major bacterial groups contain LPS as acomponent of bacterial cell wall and therefore act as potential sourceof elevated plasma LPS, which is associated with metabolic endotoxemia.These tissues play particularly important role in metabolic endotoxemiaand other metabolic diseases, since LPS causes inflammation in thesetissues, potentially leading to a cascade of adverse events includingimpaired glucose metabolism and reduced insulin sensitivity.

Example 2

Introduction

The aim of this study was to determine in mice the effect of high fatfeeding on intestinal flora, bacterial translocation and glycemia, aswell as the potential protective role of Lactobacillus acidophilus NCFM™(NCFM™) on these parameters.

Materials and Methods

Obesity and diabetes were induced on C57BL/6 male mice by an high animalfat diet (60% fat, HFD). Control mice received standard diet (4% of fat,LFD). From 19 weeks old, these mice received 10⁹CFU/day of Lactobacillusacidophilus NCFM™ during 3 weeks. Fasting glycemia was measured beforeand after treatment with NCFM™.

An intraperitoneal insulin sensitivity test was performed the day beforethe sacrifice. Analysis of colonic adherent flora and bacterialtranslocation was done by the following methods.

Bacterial Analysis

After dissection colon and ileum were harvested. Ileal and colon wallwas used to identify and quantify the adherent flora.

Samples were conserved up to 2 hours in Ringer/Tween 80 medium underanaerobic conditions. After homogenisation, samples were incubated inBrain Heart Broth to enrich and identify the highest number of anaerobesand aero-anaerobes bacteria.

1 in 10 and 1 in 100 dilutions were performed in Ringer cysteine medium.These dilutions were spread out on Columbia agar enriched with 5%defibrinated horse blood (CS) and incubated at 37° C. under aerobe oranaerobe atmosphere. Colonies were counted and identified 2 and 7 daysafter spreading.

Three types of medium were used. Some unselective agar, CL agar(Deoxycholate, Citrate, and Lactose) to isolate enterobacteria and MRSagar (Man, Rogosa, Sharpe) specific for Lactobacillus spp.Identification of the bacteria was performed by the determination of therespiratory type, the Gram staining and the metabolic features.

Bacterial Translocation

After dissection, mesenteric lymph nodes (MLN) and mesenteric adiposetissue (MAT) were harvested. Bacterial translocation was quantified inMLN and MAT.

Samples were conserved up to 2 hours in Ringer/Tween 80 medium underanaerobic conditions. After homogenisation, 1 mL of the initial sampleswas inoculated in brain-heart enrichment broth in order to diminish thedetection threshold for samples with a low bacterial level. Enrichmentcultures were covered by a layer of paraffin in order to allow growth ofanaerobes incubated for four days and checked for growth daily.

Positive cultures were isolated on modified Columbia blood agar platesincubated 48 h under anaerobic conditions. Different types of colonieswere subcultured and identified. All incubations were done at 37° C.Bacterial counts were established and expressed as log CFU/g of tissue.

Results

At 18 weeks old, high fat diet mice (HFD) weighed 39% ore than controldiet mice (LFD) (39.2±3.3 vs 28.1±1.1 g, p<0.01). Fasting hyperglycemiaincreased by 77% (174±31 vs 98±6 mg/dl, p<0.01), and insulin sensitivitydecreased (2 vs 48% of glycemia decrease 60 min after insulin injection)were found in HFD group compared with LFD.

Lactobacillus acidophilus NCFM™ administration did not modify fastingglycemia of LFD mice (95±5 mg/dl vs. 92±6 mg/dl). However, glycemia ofHFD mice was significantly decreased, which trended to normalization(226±23 mg/dl vs 125±10 mg/dl, p<0.02). HFD mice treated with NCFM™remained resistant to insulin.

HFD mice showed colonic adherent flora significantly more abundant(6.5±0.7 vs 4.6±0.4 logCFU/g, p<0.01) including more Lactobacilli(6.0±0.5 vs 4.5±0.2, p<0.01) and Enterococci (5.4±0.4 vs 4.4±0.2logCFU/g, p<0.01). This excess was canceled by NCFM™ treatment (back to5.4±0.6 logCFU/g for total flora, 4.3±0.2 logCFU/g for Lactobacilli, and4.6±0.4 logCFU/g for Enterococci). The increase of bacteria detected inmesenteric fat of HFD mice (3.8±1.5 vs 2.2±0.9 logCFU/g) was slightlymodified by NCFM™ treatment (4.0±1.2 logCFU/g). In colon and ileum ofHFD mice, flora diversified with appearance of Gram-negative bacteria,which were not cleared by NCFM™. In mesenteric lymph nodes,Gram-positive bacteria appeared on high fat diet mice, and disappearedafter NCFM™ treatment.

Conclusion

Probiotic strain Lactobacillus acidophilus NCFM™ tended to normalizefasting hyperglycemia of obese and diabetic mice. The latter showedadherent intestinal flora more abundant and more diversified thancontrol mice. Treatment with Lactobacillus acidophilus NCFM™ led todecrease of mucosa-adhered bacteria levels in the colon, and toclearance of potentially pathogenic Gram-positive bacteria in ileum,colon and mesenteric lymph nodes.

Example 3

Measurement of Mucosa-adherent and Luminal Escherichia coli and E. coliTranslocation

Effect of probiotic treatment on mucosal adhesion and bacterialtranslocation of E. coli was determined by culturing. The mouse modeland the probiotic treatment used were the same as described in Example 1above. In addition, a group receiving a combination of the probioticwith a prebiotic component (fructo-oligosaccharides at 0.2 g per animalper day) was included.

Fasted mice were gavaged with 10⁹ colony forming units of ampicillinresistant E. coli isolated from the mouse and rendered ampicillinresistant by the mean of a plasmid expressing the β-lactamase gene underthe control of a prokaryotic promoter. Two hours later the mice weresacrificed and the mucosa or the lumen from the duodenum, the jejunum,the ileum, the caecum and the colon, were harvested separately, diluted,plated onto ampicillin-containing agarose and incubated overnight at 37°C. The number of colonies was counted. Similarly, ampicillin resistantfluorescent E. coli was cultured from harvested tissues including liver,spleen, mesenteric adipose tissue, subcutaneous adipose tissue,mesenteric ganglion and subcutaneous ganglion. Insulin concentration wasmeasured from plasma in fasted state as well as in fed state.

Results

In order to investigate the role of mucosal adhesion in this process,the adherence of antibiotic-resistant commensal Escherichia coli intothe mouse intestinal mucus was quantified. The quantification ofantibiotic resistant colonies on an agarose plate originating from themucosa or different segments of the lumen showed that the HFD dietelevated the levels of mucosa-associated E. coli, but treatment withprobiotic strain B420 reduced the HFD-induced E. coli mucosal adherenceto the jejunum and the ileum (FIG. 7). Although initial tests revealedno changes in the caecum or colon, further tests showed that probioticstrain 420 reduced the HFD-induced E. coli mucosal adherence to thecaecum (FIG. 8).

Treatment with B420 alone or in combination with a prebiotic alsoreduced the translocation of E. coli into mouse tissues includingtissues including liver, spleen, mesenteric adipose tissue, subcutaneousadipose tissue, mesenteric ganglion and subcutaneous ganglion (FIG. 9).Reduction of mucosal adhesion and translocation of E. coli wasaccompanied with improved insulin levels, namely, reduced fed insulinlevels and improved insulin secretion upon feeding (FIG. 10).

Example 4

Reduction of Gene Expression

Materials & Methods

Caco-2 cells (ECACC, Salisbury, UK) were used as a model for humanintestinal epithelial cells (IECs) as in Putaala H, et al. (“Effect offour probiotic strains and Escherichia coli O157:H7 on tight junctionintegrity and cyclo-oxygenase expression”, Research in Microbiology2008) and Makivuokko H, et al. (Nutr. Cancer, 2005, 52, 94-104). instandard in vitro culture conditions comprising a humified athmosphereat 37° C. with 5% CO₂, and Caco-2 cell culture media (Invitrogen,Carlsbad, Calif., US and BD Biosciences, San Jose, Calif., US).

For exposure of Caco-2 cells Bifidobacterium animalis ssp. lactis 420was propagated in Man, Rogosa and Sharpe (MRS) broth overnight,bacterial cell densities determined with flow cytometry (FACS Calibur,Becton Dickinson, San Jose, Calif., US), pelleted (25° C., 5 min, 3000g), washed once with Caco-2 culture medium, and diluted into Caco-2culture medium in a ratio of 20 to 100 bacterial cells to one Caco-2cell. After this the Caco-2 cells were treated for 8 hours in standardin vitro conditions. As a control, Caco-2 cells treated with Caco-2culture medium without added B. animalis ssp. lactis 420 were used.

For exposure of Caco-2 cells with polydextrose, 1% (v/v) and 2% (v/v)polydextrose (Litesse®) in colon simulation medium were first fermentedin an in vitro colon simulator as in Makivuokko et al. referred toabove. The in vitro colon simulation consisting of four stages and offour parallel units was used in this study.

A single unit comprises four sequentially connected vessels (V1, V2, V3and V4) with conditions adjusted to represent different parts of thehuman colon in series representing different parts of colon: proximal,ascending, descending, and distal colon. After simulation thepolydextrose fermentation supernatants were pelleted (25° C., 5 min,10,000 g), diluted 10% (v/v) into Caco-2 culture medium. The cells weretreated for 24 h in standard in vitro conditions. As a control, Caco-2cells treated similarly with fermented colon simulation medium withoutadded polydextrose were used.

After exposure, total cellular RNA was isolated from Caco-2 cells usingRNeasy mini kit (Qiagen, Hilden, Germany). Affymetrix U133+2.0 GeneChips(Affymetrix Inc. Santa Clara, Calif., US) were used to study the humancell transcriptome. All RNA processing, labelling, hybridizations,washing, staining and scanning during the microarray processing wereperformed according to the manufacturer's standard recommendations.

Microarray data was pre-processed with GC-RMA, and analyzedstatistically using programming language R (version 2.5.0) (RDevelopment Core Team; “R:A language and environment for statisticalcomputing”, Vienna, Austria: R Foundation for Statistical Computing;2006), and Bioconductor (version 2.0) (Gentleman R C et al.“Bioconductor: open software development for computational biology andbioinformatics” Genome Biol. 2004, 5, R80). Differentially expressedgenes were selected based on gene expression with more than 1.6log-ratio difference in the signal intensity compared with that ofcontrol for samples treated with B. animalis ssp. lactis 420. Forpolydextrose treated Caco-2 samples differentially expressed genes wereselected based on their total behaviour calculated using the norm ofgenes from the three last vessels (V2, V3, V4) of simulation.

Most of the genes followed similar pattern across the four vessels:within down-regulated or up-regulated profiles the expression decreasedor increased towards the last vessel. Also, if a gene profile wasup-regulated in control, 0% polydextrose, it was slightly moreup-regulated in 1% polydextrose treatment and even more in 2%polydextrose. Similarly, in down-regulated profiles, the 2% polydextrosetreatment was more down-regulated than 1% which was more down-regulatedthan control. Thereby, genes were defined as either up- ordown-regulated based on the last three vessels. The difference betweenthe non-treated samples and two polydextrose concentrations wascalculated using Student's t-test for paired samples with cut-offp-value p<0.01.

Results

Expression of intestinal cholesterol absorption-related genes isregulated in Caco-2 cells by B. animalis ssp. lactis 420 (Table 1) or bypolydextrose fermentation supernatants (Table 2).

The downregulative effect on APOB, APOC2, APOC3, APOAIV and MTTP havereductive effect on cholesterol and triglyceride absorption from theintestine (Iqbal J and Hussain M M, American Journal ofPhysiology-Endocrinology and Metabolism 2009, 296, E1183-E1194), whereasupregulative effect on NPC1 increases the formation of HDL in theintestine (Branham L R et al, J Clin Invest 2006, 116, 1052-62). NR2F2and HNF4A are transcription factors that regulate the expression ofapolipoprotein genes: see Antes T J, et al. Biochemistry 2101, 40,6720-30 and Leng S Y, et al. American Journal ofPhysiology-Gastrointestinal and Liver Physiology, 2007, 293, G475-G483.

TABLE 1 Effect of Bifidobacterium animalis ssp. lactis 420 GeneExpression level Apolipoprotein B (APOB) −2.89 Apolipoprotein C-II(APOC2) −2.53 Apolipoprotein C-III (APOC3) −1.47 Apolipoprotein A-IV(APOAIV) −2.84 Microsomal triglyceride transfer protein (MTTP) −2.99Nuclear receptor subfamily 2, group F, member 2 −1.67 (NR2F2), alsoknown as ARP1 Hepatocyte nuclear factor 4, alpha (HNF4A) −2.3

TABLE 2 Effect of polydextrose fermentation Expression level 1% 1% 1% 1%2% 2% 2% 2% V1 V2 V3 V4 V1 V2 V3 V4 Niemann-Pick 1.03 1.87 1.62 1.681.20 1.82 1.99 1.78 disease, type C1 (NPC1)

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the following claims.

The invention claimed is:
 1. A method of treating metabolic endotoxemiain a mammal in need thereof, the method comprising administering aneffective amount of a bacterium selected from a lactic acid bacterium, aBifidobacterium or a mixture of any thereof, wherein the metabolicendotoxemia is induced by a high fat diet.
 2. A method according toclaim 1, wherein the endotoxemia comprises an increase of the level oflipopolysaccharides in the mammal by a factor in the range from 2 to 4compared with basal lipopolysaccharide levels.
 3. A method according toclaim 1, wherein the bacterium is a probiotic lactic acid bacteriumand/or a probiotic Bifidobacterium.
 4. A method according to any claim1, wherein the bacterium is a bacterium selected from the generaLactobacillus or Bifidobacterium and mixtures thereof.
 5. A methodaccording to claim 4, wherein the bacterium is selected from the speciesLactobacillus acidophilus, Lactobacillus plantarum, Bifidobacteriumanimalis, Bifidobacterium lactis, or Bifidobacterium bifidium, andmixtures thereof.
 6. A method according to claim 5, wherein thebacterium is Lactobacillus acidophilus strain NCFM, Bifidobacteriumanimalis subsp. lactis strain 420, Lactobacillus salivarius strain 33,or a mixture thereof.
 7. A method according to claim 1, wherein themammal is diabetic and obese.
 8. A method according to claim 1, whereinthe mammal is diabetic and non-obese.
 9. A method according to claim 1,wherein the mammal is non-diabetic and obese.
 10. A method according toclaim 1, wherein the mammal continues to ingest a high-fat diet duringthe course of the treatment.
 11. A method according to claim 1, whereinthe bacterium is administered in combination with one or moreprebiotics.
 12. A method according to claim 11, wherein the prebiotic isselected from alginate, xanthan, pectin, locust bean gum, inulin, guargum, a galacto-oligosaccharide, a fructo-oligosaccharide, polydextrose,lactitol, lactosucrose, a soybean oligosaccharide, palatinose, anisomalto-oligosaccharide, a gluco-oligosaccharide or axylo-oligosaccharide, or a mixture of any thereof.
 13. A methodaccording to claim 12, wherein the prebiotic is polydextrose.
 14. Amethod according to claim 1, wherein the endotoxemia comprises anincrease of the level of lipopolysaccharides in the mammal by a factorin the range from 1.5 to 20 compared with basal lipopolysaccharidelevels.